Study on structural characteristics, physicochemical properties, and in vitro digestibility of Kudzu‐resistant starch prepared by different methods

Abstract Three different methods, including autoclaving, autoclaving–debranching, and purification, were used to prepare Kudzu‐resistant starch (KRS) from Kudzu starch (KS). The physicochemical properties, such as thermodynamic properties, pasting properties, solubility, swelling, and coagulability, as well as the in vitro digestive characteristics of the three kinds of KRS were studied. The results showed that the morphology of starch granules of KRS prepared by autoclave, autoclave enzymatic hydrolysis, and purification methods was changed and the relative crystallinity was significantly decreased compared with the original starch. X‐ray diffraction (XRD) showed that KRS exists in the form of C and C+V crystalline form. There was a significant increase in the pasting temperature and a remarkable decrease in the peak viscosity and the expansion degree of the KRS prepared by all three methods. The solubility of the resistant starch (RS) obtained by autoclaving–debranching and that by purification were both increased compared to that of native KS, while the solubility of the RS obtained by autoclaving was decreased. Meanwhile, the retrogradation of the three RS was also improved to varying degrees. The contents of RS in the samples were: P‐KRS (71%) > DA‐KRS (43%) > A‐KRS (42%) > KS (9%). Simulated human in vitro digestion experiments showed that RS has stronger antidigestibility properties than native starch. Among them, the RS prepared by the purification method has stronger antidigestive properties, and it is predicted that it may have a better potential value in regulating blood glucose. These results indicated that the processing properties of KRS, especially the digestibility, are significantly improved and can be used as a new functional food ingredient, which deserves thorough study.


| INTRODUC TI ON
Kudzu (Pueraria lobata) is considered a classic medicinal food homologous plant, widely distributed in the countries of East Asia and America. Kudzu has been reported to have various healthmaintaining pharmacological functions, including antidiabetic activity, antihyperlipidemic activity, and prevention of cardiovascular diseases, due to numerous active components such as isoflavones, saponins, polysaccharides, and starch (Duru et al., 2020;Penetar et al., 2015;Wang, Zhu, et al., 2017;Yang et al., 2016). Kudzu has been widely used in Chinese medicine and food production. Kudzu contains 53% ~ 66% carbohydrate and is mostly in the form of starch. Modern research on Pueraria lobata has mainly focused on small-molecule active ingredients, and large amounts of starch have been discarded after large-scale extraction, resulting in a waste of resources.
According to different classification methods, starch can be divided into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) (Englyst et al., 1992). RS, a product of starch digestion, is poorly absorbed by the small intestine and has a beneficial effect on human health (Englyst & Cummings, 1985). RS has received a lot of attention because of its physiological functions similarly to those of the traditional dietary fiber. Previous studies have shown that RS has many beneficial effects on human health, including the prevention of obesity, diabetes, and colon cancer (Bindels et al., 2017;Birt et al., 2013;Giacco et al., 2014;Zhang et al., 2020).
Researchers have been dedicating their efforts to the preparation of RS because of its ability to offer a broad range of applications. Methods for preparing RS include heat treatment, enzyme treatment, combined heat and enzyme treatment, and chemical treatment. Heat treatment has the advantages of simple operation, low cost, high yield, no pollution, etc. (Sajilata et al., 2006). The enzyme treatment method not only has the above advantages, but also has strong enzyme specificity. Nevertheless, chemical treatment is less used in food because of the poor debranching effect, low yield, and low safety. Ultrasonication is an emerging technology that is efficient, environmentally friendly, safe, and easy to apply, but has high production costs (Huang et al., 2016). Guo et al. prepared Kudzu-resistant starch (KRS) using α-amylase and glucoamylase and found that the contents of RSD, SDS, and RS were 5.66%, 25.88%, and 68.46%, respectively, and the crystallinity increased from 34.8% to 43.6% with the increase of enzymatic digestion time (Guo et al., 2016). Feng et al. produced KRS with better water-holding capacity and transmittance than native starch by debranching and subsequent recrystallization and the content of RS was 45.4% (Zeng et al., 2019).
The KRS prepared by the annealing method by Zhu et al. showed an increase in RS content compared to Kudzu starch (KS) content but still very low (2.51% ~ 8.59%) (Zhu et al., 2018). Some chemical modification methods, such as hydrochloric acid hydrolysis, acid hydrolysis-hydroxypropylation, carboxymethylation, cross-linking, and esterification, were also applied to prepare KRS, but these methods have poor safety Li et al., 2012;Tang et al., 2012;Wang et al., 2010;Zhao et al., 2017). Nevertheless, these methods have problems such as complicated preparation process, poor safety, and low RS content, so we will use a simpler and safer method to prepare KRS. Up till now, limited articles are available on the structural characterization of KRS in different treatments and the relationship between digestion resistibility and structure.
In recent years, the demand for nutritious and healthy food has become more and more urgent with the evolution of dietary structure and disease spectrum. In this study, KRS was prepared from Kudzu by autoclaving, autoclaving-debranching, and purification.
The purpose of this study was to compare the structural characteristics, physicochemical properties, and digestive characteristics of KS and KRS to lay the foundation for further studies on the blood glucose regulation function of KRS.

| Preparation of KRS
Autoclaving Kudzu-resistant starch (A-KRS): The KS suspension (12%, w/w, dry-base starch) was heated at 90°C in a water bath for 30 min. The gelatinized starch paste was heated at 121°C for 30 min, cooled at room temperature for 1 h, and refrigerated at 4°C for 12 h. Finally, the starch obtained was dried to a constant weight at 60°C and ground through a 100-mesh sieve to obtain the resistantenhanced A-KRS.
Autoclaving-debranching Kudzu-resistant starch (DA-KRS): Following the autoclave method, take 100 g of KS and allow to cool at room temperature after the autoclave has been completed. Citrate buffer (0.135 mol/L) was used to adjust the pH to 4.5, followed by the reaction between prulanase (60 U/g) and suspension at 40°C for 4 h. After that, the starch suspension was inactivated at high temperature and cooled to room temperature, then the prulanase was removed by centrifugation at 892g for 10 min. Later, the mixture was allowed to age for 24 h under refrigeration, dried at 60°C for 12 h, and then crushed and ground through a 100-mesh sieve to obtain the DA-KRS.
Purification Kudzu-resistant starch (P-KRS): After autoclavingdebranching, the treated starch was prepared with citric acid buffer (0.135 M, pH = 2). Then, 10 ml of pepsin (400 U/ml) was added to 15% starch milk, stirred in a water bath at 40°C, and adjusted the pH to 7, then 10 ml of high-temperature-resistant α-amylase (400 U/ml) was added, stirred in a water bath at 95°C for 30 min, adjusted the pH to 4.5, and added 0.6 g of glycosylase (100,000 U/g), and stirred in a water bath at 60°C for 1 h. The enzyme was inactivated, and the supernatant was removed after repeated washing with 95% ethanol, stirred in the water bath at 60°C for 1 h. After repeated washing, the supernatant was removed, dried at 60°C for 12 h, crushed, and sieved, and the P-KRS was obtained.

| Surface morphology and granule size
The morphology of the dried starch samples was examined using a Quanta 250 FEG scanning electron microscope (SEM) (FEI Ltd., Oregon, USA) with an accelerating voltage of 15 kV .
The dried starch materials were sprayed on circular metal stubs previously covered with double-sided adhesive and coated with gold prior to SEM examination.
The granule sizes of KS and KRS were analyzed using a laser diffraction particle size analyzer (Mastersizer 2000, Malvern). For laser diffraction analysis, starch granules were suspended in distilled water and stirred. A dry method was used with a particle refractive index of 1.55. Duplicate measurements were carried out for each of the starch preparations. Particle size was obtained and expressed in terms of the mean particle size, D (0.1), D (0.5), D (0.9), D (3, 2), and D (4, 3).

| Fourier transform infrared (FT-IR) spectroscopy
The FT-IR spectra of KS were obtained with an FT-IR spectrometer (PerkinElmer, USA) using a transmission mode. The starch sample (2 mg) was blended with potassium bromide (KBr) powder (150 mg) by grinding for 10 min under the infrared lamp and pressed into tablets before measurement. Each spectrum was recorded at a resolution of 4 cm −1 and at room temperature with 16 scans. Spectra were corrected and then deconvoluted in the range of 4000 ~ 400 cm −1 (Zheng et al., 2016).

| X-ray diffractometry (XRD)
The starch samples were analyzed using X-ray diffractometer (Bruker Corp, Germany) operating at 40 KV and 40 mA at diffraction angle 2θ of 4° to 40° with step intervals of 0.02° at a scanning speed of 4°/min. The XRD data were analyzed by Origin, and the relative crystallinity was calculated by the following formula (Nara & Komiya, 1983): where A c and A a stand for the crystal and amorphous areas, respectively.

| Differential scanning calorimetry (DSC)
Thermal properties of KS were investigated with the use of a differential scanning calorimetry (DSC-7000X, HITACHI, Japan). The dried starch sample was thoroughly mixed with distilled water in a ratio of 1:3. The pans were sealed, equilibrated for 24 h at room temperature, and then heated from 20 to 180°C at the rate of 10°C/min. An empty pan was used as a reference. Onset temperature (T 0 ), peak temperature (T p ), conclusion temperature (T c ), and enthalpy values (∆H) were measured from the DSC thermogram (Zeng et al., 2016).
Add 25 ml of distilled water into starch samples (3 g of total weight), then increase the temperature from 50°C to 95°C, and the whole procedure takes 13 min until the end .

| Solubility and swelling power
Starch suspension (1%) was heated at 55°C, 65°C, 75°C, 85°C, and 95°C in a water bath for 30 min and then cooled to room temperature. The suspension was centrifuged at 3000g for 10 min (TGL-200MC, Hunan Xiangxi Instrument Co., Ltd., China). The supernatant and the sediment were separated, an aliquot of the supernatant was evaporated at 110°C, and the swollen starch sediment was weighed (Li et al., 2020). Solubility and swelling power were calculated as follows: where S is the solubility power, A is the weight of dried supernatant (g), W is dried starch weight (g), P is the swelling power, and B is the weight of sediment (g).

| Condensation
Starch suspension (1%) was heated at 95°C for 30 min in a water bath, cooled to room temperature, and then poured it into a graduated cylinder and let it settle. Carefully observe the retrogradation of different starch samples by the ratio of the volume of starch × 100 % paste supernatant to the total suspension at different times .

| In vitro starch digestibility
The in vitro starch digestibility was determined according to the literature, with minor modifications (Englyst et al., 1996). Briefly, 200 mg of starch sample was precisely weighed and dispersed in 0.1 mol/L sodium acetate solution, the system temperature was maintained at 37°C after homogenization. Then 2.5 ml of α-amylase (580 U/ml) solution and 2.5 ml of glycosylase (30 U/ml) solution were added. The enzymatic hydrolysate (0.2 ml) at 20 and 120 min was removed and boiled for 5 min to deactivate the enzymes, and then centrifuged at where G 20 and G 120 indicate the glucose content (mg) released after 20 and 120 min, respectively, and TW is the total starch weight (mg). Goñi et al. (1997a) method was adopted with slight improvements.

| Kinetics of in vitro enzymatic hydrolysis of starch
Draw the hydrolysis curve of different starch samples at 10, 20, 30, 40, 60, 90, 120, 180 min, and perform first-order kinetic fitting of the hydrolysis curve Shi & Gao, 2011). The formula is as follows: The area under the hydrolysis curve (AUC) was calculated using the following equation: where C ∞ is the equilibrium concentration (%), k is the reaction kinetic constant (min −1 ), t f is reaction termination time (180 min), and t 0 is initial reaction time (0 min).
The area of white bread under the hydrolysis curve (0 ~ 180 min) was used as the standard, and the HI (%) was calculated as the percentage of the integral area of the hydrolysis curve released from the sample compared to that of white bread. The GI (%) of the samples was estimated as follows:

| Statistical analyses
All analyses were carried out in triplicate, and the values were described as the means ± standard deviations. Data were subjected to the analysis of variance (ANOVA) by SPSS, and the significance level was determined at the 95% confidence level (CI) (p < 0.05).

| Surface morphology and granule size
The average particle size of native KS was small, with d (0.5) of 8.54 ± 0.09 μm and d (0.9) of 17.95 ± 0.90 μm, as shown in Table 1.
The particle size distribution of different starch samples is shown Results are expressed as mean ± standard deviation. d (0.1), d (0.5), and d (0.9) are the particle diameters with volume fractions of 10%, 50%, and 90%, respectively. D (3,2) is the average particle size of the surface area, and D (4,3) is the volume average particle size. Values followed by different superscripts in the same column are significantly different (p < 0.05).

F I G U R E 1 Particle size distribution of different starch samples
in Figure 1. According to the results, the particle size of native KS appears as two peaks, indicating that the particle size distribution is not uniform and there are obvious particle size differences. The three kinds of KRS have a larger particle size range and particle size than the native KS, and the volume distribution is complete, showing an obvious unimodal curve, indicating that part of the KS particles swelled, aggregated, or crystallized repeatedly during the process of forming the RS. In addition, the single peak of A-KRS is sharper than that of DA-KRS. The uneven particle size distribution of P-KRS indicates that the granule texture is tight after multiple enzymatic hydrolyses, and the degree of recrystallization is reduced.

| Granule morphology
The

| FT-IR analysis
Infrared spectroscopy was used to detect changes in functional groups during the formation of RS. As shown in Figure

| Crystalline structure
The XRD patterns and relative crystallinity of KS, A-KRS, DA-KRS, and P-KRS are presented in Figure 4. All samples exhibited a strong peak at 26.6°, which is typically type A crystal peak (Ma et al., 2018).  which results in a lower crystallinity of RS than KS. DA-KRS formed new crystals after debranching by prulanase, so the crystallinity was higher than that of A-KRS. The increased crystallinity of P-KRS was due to the hydrolysis of the amorphous layers of starch by the action of high-temperature-resistant α-amylase, and the amorphous more crystalline peak decreased slightly, thus the crystallinity increased (Cai & Wei, 2013), and the results were consistent with the results reported by Guo et al. (2016).

| Thermal properties
Starch gelatinization is a process of energy transfer, and the thermodynamic parameters of different starches have obvious differences. The thermodynamic parameters of different starch samples are listed in Table 2

| Pasting properties
There were different changes in the starch granules of KRS prepared by different processing processes, such as heating and enzymatic hydrolysis. Whether the starch swells during heating or maintains its stable structure after enzymatic hydrolysis has a considerable influence on its viscosity after gelatinization (Jay-lin, 2006). The pasting properties of starches analyzed using RVA are summarized in Table 3. KRS granules prepared by different methods were all more thermally stable, and all the gelatinization indexes were reduced compared to the native KS after heating into paste. DA-KRS and P-KRS had a much lower viscosity characteristic index than KS and were unable to gel below 100°C. This may be due to the fact that DA-KRS and P-KRS form a more compact crystal structure with small intermolecular gaps, which prevents the powder from punching and absorbing water to form a paste at the set temperature. Compared with DA-KRS and P-KRS prepared by enzymatic digestion, the A-KRS prepared by pressure and heat treatment exhibited higher pasting characteristics, and some characteristics, such as Valley and Final viscosities, were close to those of the original starch. This may be related to the fact that autoclaving acts mainly on the amorphous regions of the starch. During this high pressure and temperature process, the A-KRS starch molecules absorbed water and formed stable hydrogen bonds for adsorption, resulting in a significant increase in intermolecular gravitational forces, which directly leads to higher viscosities (Govindasamy et al., 1996). The addition of 5% RS to the noodles reduced the viscoelasticity of the noodles, which facilitated less chewing, increased satiety, and reduced carbohydrate intake, but had little effect on the cooking characteristics of the noodles (Punia et al., 2019). Raungrusmee et al. developed glutenfree noodles with low glycemic index using rice RS, while the addition of xanthan gum improved the texture of RS gluten-free noodles (Raungrusmee et al., 2020). Therefore, adding the right amount of RS to pasta products can make the performance and flavor of pasta products better and better meet the needs of consumers.

| Swelling power and solubility
Swelling power and solubility behavior of KS, A-KRS, DA-KRS, and P-KRS starches at different temperatures are shown in Figures 5 and   6, respectively. The solubility and swelling degree of starch are the key indicators for judging the hydrogen bond force between starch and water (Chen et al., 2015). As shown in Figure 5, with the increase of solution temperature, the water solubility of each starch sample was enhanced significantly, which was consistent with the dissolution behavior of starch in water. Overall, the RS obtained by compression heat treatment exhibited poor solubility. In contrast, the solubility of RS prepared by enzymatic treatment was significantly enhanced compared with the native starch. In particular, DA-KRS had superior solubility properties, suggesting its better potential for application in food processing. The lower water solubility of A-KRS may be related to its stable crystal structure, which provides better encapsulation of small molecules of starch. Moreover, the stable crystal structure makes it difficult for the starch to absorb water, which is manifested by the difficulty of swelling effect, as shown in Figure 6. While DA-KRS and P-KRS had undergone enzymatic treatment, the structure of the microcrystalline bundle of starch molecules was loosened, and the starch molecules could easily escape with water under high-temperature conditions (Barua et al., 2021;Fang et al., 2021). P-KRS was purified after enzymatic digestion, and most of the soluble sugars and small molecules were removed, resulting in higher crystallinity but significantly lower solubility and swelling power.

| Condensation
The condensation of starch reflects the ability of starch molecules to hydrogen bond with water (Biduski et al., 2018). As shown in were more significant after binding with water, as well as its gel density after gelation was close to that of water. Accordingly, the precipitation speed is slower, appearing as a smaller supernatant volume, as shown in Figure 7. The molecules of A-KRS, DA-KRS, and P-KRS were highly helical and had the structural characteristics of aged starch, and this structure was difficult to be destroyed when reheated. RS has low water-holding capacity and is suitable for use as a quality improver in low-moisture foods, such as bread, cookies, cakes, etc. Cross-linked wheat RS with low water-holding capacity was added to the cookies to make the dough softer and to produce cookies with high fiber and low glycemic index, and the width and thickness values of the cookies were significantly increased and the quality was significantly improved (Kahraman et al., 2019). The addition of RS to solid beverages and jams can enhance the stability and textural properties and keep them unstratified for a long time (Gao et al., 2021).

| In vitro starch digestibility
Digestion of starch granules is a complex process, which is influenced by the surface characteristics and internal structure of the starch granules and the starch crystal structure, as well as the interaction of many factors, such as starch source, straight-chain starch content, and relative crystallinity (Ambigaipalan et al., 2011). RDS, SDS, and RS contents of KS, A-KRS, DA-KRS, and P-KRS are presented in Figure 8. KS was contained in 81% RDS, 11% SDS, and 9% RS, but the high content of RDS leads to a high glycemic index, which is harmful to health. After autoclaving, the content of RDS decreased while RS increased significantly. This change might be caused by the reduction of enzyme contact sites due to the destruction of starch crystal structure. The different starch ratios of P-KRS followed the order: 71% RS > 29% RDS > 1% SDS. The higher RS content in P-KRS was attributed to short amylose molecule that recrystallizes to form a dense structure. Amylase has removed excess small molecules of sugar, thus increasing the RS content. As a functional food, RS has been given great importance benefit for decreasing the incidence of enteric disease. Moreover, RS is a good substrate for butyrate production, which can alter microRNA (miRNA) levels in colorectal F I G U R E 7 Supernatant of different starch samples cancer cells to reducing risk associated with a high red meat diet (Humphreys et al., 2014). In addition, a great number of RS is used as food additive, thereby giving favorable brittleness and tenderness to bread crumbs (Djurle et al., 2018).

| Kinetics of in vitro enzymatic hydrolysis
The in vitro enzymatic hydrolysis rates of KS, A-KRS, DA-KRS, and P-KRS are shown in Figure 9. The trends in hydrolysis rates for the types of starch and white bread controls were relatively similar. The hydrolysis rate was fast during the first 10 min at the beginning of digestion, and after 10 min, the degree of hydrolysis increased slowly  (Dhital et al., 2010). The lowest digestibility of P-KRS was attributed to the complexation of starch with lipids to form V-shaped crystals, which inhibited the water absorption and swelling of starch granules and reduced the binding sites between starch and enzyme molecules, resulting in enzyme resistance (Crowe et al., 2000). The short interval of starch digestion kinetics monitoring can accurately reflect the changes in the middle and late stages, and can more accurately quantify the degree of digestion of each part of starch. The digestion rate may be more suitable for practical situations (Goñi et al., 1997b).
The digestive kinetic parameter of different starch samples is listed in

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets presented in this study are included in the article.